1. Volume 9, Issue 6, Pages 1227-1240 (June 2002)
Molecular Mechanism for the Regulation of Protein Kinase B/Akt by Hydrophobic Motif Phosphorylation 
Jing Yang, Peter Cron, Vivienne Thompson, Valerie M. Good, Daniel Hess, Brian A. Hemmings, David Barford 
Molecular Cell 
Volume 9, Issue 6, Pages (June 2002)
DOI: /S (02)

2. Figure 1 Structure of PKB and Comparison with PKA
Ribbons representation of PKA (A) and PKB (B). Catalytic and regulatory structural elements are color coded, with αB and αC helices gold, DFG and APE motifs of the activation segment red, remainder of activation segment yellow, catalytic loop gray, β1/β2 glycine-rich loop pink, and hydrophobic motif of PKA gray. PKA and PKB were superimposed onto their C-terminal lobes. Phe 294 of the DFG motif of PKB occupies a site equivalent to the adenine pocket of the nucleotide binding site of PKA. (C) Stereo view of a superimposition of PKA and PKB to show different relative orientations of their N- and C-terminal lobes. Main chain coil of PKA is colored as in (A). PKB is colored green. Conformational differences in C lobe are localized to the activation segment and αF/αG loop. (D) Schematic of PKB. Figure drawn using BOBSCRIPT (Esnouf, 1997) and RASTER3D (Merit and Murphy, 1994)
Molecular Cell 2002 9, DOI: ( /S (02) )

3. Figure 2 Structure of the N-Terminal Lobe
(A) Flexibility of αB and αC helices. 2Fo-Fc electron density map contoured at 1σ of a portion of the N-terminal lobe of pΔPH-PKB-ΔC (β3, β4, β5 strands, αB and αC helices). Electron density for the β sheet is well resolved, whereas the αB and αC helices are disordered. Main chain and side chain of PKB residues are shown as coil in yellow and atom bonds, respectively. The main chain of the N-terminal lobe and hydrophobic motif of PKA is shown in blue and superimposed onto PKB.
(B and C) Role of hydrophobic motif to order the αB and αC helices and link to activation segment. (B) Interactions of hydrophobic motif of PKA with the β5 strand and αB and αC helices of the N-terminal lobe. Phe 347 and Phe 350 are buried by hydrophobic residues. Glu 349 and C-terminal carboxylate form hydrogen bonds with basic residues of the αC helix. (C) Disorder of the αB and αC helices of PKB is correlated with absence of bound hydrophobic motif. Residues mutated to test responsiveness to PIFtide (Figure 7B) are colored pink. In (B) bracketed residues correspond to PKB numbering
Molecular Cell 2002 9, DOI: ( /S (02) )

4. Figure 3
Role of αC Helix to Regulate Conformation of PKA and PKB and Structure of Activation Segment and DFG Motif
(A) αC helix stabilizes an active state of PKA by interaction with pThr 197 of the activation segment via His 87, and Phe 185 of the DFG motif via Ile 93 and Leu 94.
(B) In PKB, disorder of the αC helix prevents His 196 from interacting with pThr 309; Phe 294 of the DFG motif binds within the nucleotide binding site of ATP.
Molecular Cell 2002 9, DOI: ( /S (02) )

5. Figure 4 Features of the Hydrophobic Groove
(A) Conservation of hydrophobic motif binding channel among AGC kinases. The molecular surface of PKA is calculated with residues omitted and is color coded according to sequence conservation with color ramped from red (invariant) to blue (nonconserved). Kinase sequences used to determine conservation are PKBβ, PKA, PKC, p70-S6K, p90-S6K, PDK1, SGK, and NDR1. Residues of the hydrophobic motif (Phe 347 to Phe 350) of PKA are shown. Figure drawn using GRASP (Nicholls et al., 1991).
(B) Electrostatic potential of the hydrophobic groove.
Molecular Cell 2002 9, DOI: ( /S (02) )

6. Figure 5 Multiple Sequence Alignment of Various AGC Kinases
Invariant residues are colored red, conserved are yellow. The positions of critical functional residues are indicated with a blue arrow and numbered according to PKA. PKB Thr 309 and Ser 474 phosphorylation sites are indicated. The conserved AGC kinase hydrophobic motif is shown and mutated residues of PKB that influence PIFtide activation (Figure 7B) are indicated by gray arrows. Figure drawn using ALSCRIPT (Barton, 1993).
Molecular Cell 2002 9, DOI: ( /S (02) )

7. Figure 6
Activation of PKB by Hydrophobic Motif Peptide and PIFtide and Complex Formation between PKB and PIFtide
(A) Dose response curve for the stimulation of pΔPH-PKB-ΔC kinase activity by various PKB HM peptides, a synthetic 23 residue peptide encompassing the PKB HM motif. Closed circles, phosphorylated peptide; closed triangles, S474D mutant peptide; open circles, unphosphorylated peptide.
(B) Dose response curve for the stimulation of (p)ΔPH-PKB-ΔC kinase activity by PIFtide, a synthetic 24 residue peptide encompassing the PRK2 HM motif. Closed circles, PIFtide and pΔPH-PKB-ΔC; closed triangles, PIFtide and ΔPH-PKB-ΔC; open circles, mutant PIFtide(D>A) and pΔPH-PKB-ΔC. The maximal activity of PIFtide stimulated ΔPH-PKB-ΔC is 350 nmol/min/mg, equivalent to Thr 309 and Ser 474 phosphorylated ΔPH-PKB.
(C) Isothermal titration calorimetry measurements of the binding of PIFtide to pΔPH-PKB-ΔC (left) and ΔPH-PKB-ΔC (right). Upper panel, raw data of the titration of PIFtide into (p)ΔPH-PKB-ΔC. Lower panel, integrated heats of injections, corrected for the heat of dilution, with the solid line corresponding to the best fit of the data using the MicroCal software.
Molecular Cell 2002 9, DOI: ( /S (02) )

8. Figure 7
Conserved Residues of the Hydrophobic Motif, and Residues of the N Lobe of PKB, Are Required for PIFtide and PKB HM Peptide-Mediated Stimulation of PKB Kinase Activity
(A) Mutations of conserved hydrophobic motif residues of PIFtide and PKB HM peptide reduce or eliminate their potential to activate ΔPH-PKB-ΔC phosphorylated on Thr 309.
(B) Mutations of hydrophobic and electrostatic residues of the ΔPH-PKB-ΔC N lobe hydrophobic groove reduces the stimulation of PKB activity by 130 μM PIFtide. The positions of mutated residues on PKA and PKB (R202D, V194A-V198A, and L225A) are shown colored pink in Figures 2B and 2C and shown in Figure 5.
Molecular Cell 2002 9, DOI: ( /S (02) )